Thiamin is an essential cofactor in all living systems and is a required component of the human diet. Short term deprivation results in beri beri and Wemicke's encephalopathy and prolonged deprivation is lethal. Thiamin is also an important commercial chemical;it is widely used as a food additive and as a flavoring agent and annual production is on the order of 3,300 tons. Thiamin pyrophosphate, the active form of vitamin B1, plays an important role in carbohydrate metabolism and in branched-chain amino acid metabolism where it stabilizes acyl carbanion intermediates. Thiamin biosynthesisis not yet well understood and the reconstitutionof the pyrimidine and the thiazole moieties has only recently been accomplished in a defined biochemical system. In B. subtilis, thiamin pyrophosphate is synthesized from glycine, deoxy-D-xylulose 5-phosphate, cysteine and aminoimidizaole ribotide. The biosynthetic pathway is complex and uses 14 gene products. We have previously determined the structures of five thiamin biosynthetic enzymes and used these structures to support mechanistic studies. Our proposal has four specific aims. In aim 1, we will study the formation of the thiazole moiety by determining the structures of ThiF and ThiG, as well as the structures of the stable ThiSG and ThiFS complexes. The second specific aim describes structural studies on ThiC, the enzyme required for the formation of the pyrimidine moiety. In aim 3, we will study thiamin regulation and uptake by determining the structures of Tenl and TBP, respectively. The studies described in the first three specific aims will complete the structural characterization of the major bacterial thiamin biosynthetic pathway (excepting the membrane bound transport system). In the final specific aim, we will begin studies on thiamin biosynthesis in yeast, which proceeds by a very different pathway, by determining the structures of Thi4 and Thi5 - the only identified proteins involved in the biosynthesis of the thiazole and pyrimidine moieties in this organism. For all enzymes, we will also determine the structures of complexes and mutants, as needed, to understand the catalytic mechanisms. These studies will result in (1) an understanding of the biosynthesis of a vitamin required for all forms of life, (2) a mechanistic understanding of the unprecedented chemistry used for thiamin biosynthesis and (3) approaches for the construction of overexpression strains that can be used for the commercial production of thiamin by fermentation.